Spinal premotor interneurons controlling antagonistic muscles are spatially intermingled: Supplementary figures
Remi Ronzano*1, Sophie Skarlatou*2, Bianca K. Barriga*6,7, B. Anne Bannatyne*3, Gardave S. Bhumbra*4, Joshua D. Foster4, Jeffrey D. Moore8, Camille Lancelin1, Amanda Pocratsky1, Mustafa Görkem Özyurt1, Calvin C. Smith1, Andrew J. Todd3, David J. Maxwell3, Andrew J. Murray5, Samuel L. Pfaff6§, Robert M. Brownstone1§, Niccolò Zampieri2§, Marco Beato4§
1 Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK
2 Max Delbrück Center for Molecular Medicine (MDC), Roybert-Rössle-Str. 10, 13092 Berlin, Germany
3 Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, West Medical Building, Glasgow G12 8QQ, UK
4 Department of Neuroscience Physiology and Pharmacology (NPP), Gower Street, University College London, WC1E 6BT, UK
5 Sainsbury Wellcome Centre for Neural Circuits and Behaviour, University College London, London W1T 4JG, UK
6 The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla CA 92037, USA
7 Biological Sciences Graduate Program, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92037, USA.
8 Howard Hughes Medical Institute and Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, MA 02138, USA
*equal contribution
§co-senior authors
Corresponding authors:
Niccolò Zampieri, Max Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13092 Berlin, Germany: niccolo.zampieri@mdc-berlin.de
Samuel J. Pfaff, The Salk Institute for Biological Studies, Gene Expression Laboratory, 10010 North Torrey Pines Road, La Jolla CA 92037, USA: pfaff@salk.edu
Robert M. Brownstone, Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK: r.brownstone@ucl.ac.uk
Marco Beato, Department of Neuroscience Physiology and Pharmacology (NPP), Gower Street, University College London, WC1E 6BT, UK: m.beato@ucl.ac.uk
Key words: spinal cord, premotor interneurons, flexor muscles, extensor muscles, rabies, viral tracing
Abstract
Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized we used multiple complementary viral-tracing approaches to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.
Figure 3 figure supplement 1
Figure 3 figure supplement 1: The distribution of premotor interneurons is similar throughout the lumbar spinal cord: data pooled from 18 experiments (11 LG and 7 TA injections) show that within each lumbar segment, from L1 to L6, the distributions of LG and TA premotor interneurons are overlapping.
Figure 3 figure supplement 2
Figure 3 figure supplement 2: Same data as in Figure 3-figure supplement 1 shown before normalization procedures, with idealized spinal cord section scaled to the average size of each segment. Using the raw coordinates, the distribution of LG and TA premotor interneurons are consistently overlapping throughout the lumbar segments.
Figure 3 figure supplement 3
Figure 3 figure supplement 3: distribution of flexor
and extensor premotor interneurons pooled across all LG and TA
injections shown in the transverse plane (left) and as front (middle)
and lateral (right) view along the rostrocaudal axis.
Figure 4 figure supplement 1
Figure 4 figure supplement 1: distribution of flexor
and extensor premotor interneurons pooled across all LG and TA for low
titre injections shown in the transverse plane (left) and as front
(middle) and lateral (right) view along the rostrocaudal axis.
Figure 4 figure supplement 2
Figure 4 figure supplement 2: High and low efficiency infections give rise to the same premotor interneurons distributions. Comparison of high and low titre injections are shown in A and E for LG and TA respectively. For each section the data are scaled to the reference points indicated in the methods in order to account for size differences along the segments. Correlations between high and low titre experiments are high and effect sizes are low for both LG and TA muscles (B and F) The distributions are similar across experiments for both muscles (B and E) and the median values of the distributions in the ipsilateral dorsal quadrant are not different for high and low efficiency of infection (C and F).
Figure 4 figure supplement 3
Figure 4 figure supplement 3: the relation between the number of primary infected motor neurons and premotor interneurons follow a power law y = axb with a = 295 (155, 561 confidence intervals) and b = 0.53 (0.37, 0.69 confidence intervals), R2 = 0.48. Motor neuron and interneuron numbers in each experiment are shown for high (red) and low (black) titre experiments. The fitted line represents a power law.
Figure 7 figure supplement 1
Figure 7 figure supplement 1: distribution of flexor and extensor premotor interneurons pooled across GS and TA injections performed in Olig2::Cre; RΦGT mice. Distributions are shown in the transverse plane (left) and as front (middle) and lateral (right) view along the rostrocaudal axis.
Figure 12 figure supplement 1
Figure 12 figure supplement 1: Distribution of motor neurons (triangles) and premotor interneurons (dots) of 3 LG (blue) and TA (yellow) double injections (UCL) of rabies virus in ChAT::Cre+/-; RΦGT+/- mice.
Figure 12 figure supplement 2
Figure 12 figure supplement 2 Distribution of motor neurons (triangles) and premotor interneurons (dots) of 2 LG (blue) and TA (yellow) double injections (UoG) of rabies virus in ChAT::Cre+/-; RΦGT+/- mice.
Figure 12 figure supplement 3
Figure 12 figure supplement 3 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 2 LG (blue) and
MG (dark blue) double injections (UCL) of rabies virus in
ChAT::Cre+/-; RΦGT+/- mice.
Figure 12 figure supplement 4
Figure 12 figure supplement 4 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 4 LG (blue) and
MG (yellow) double injections (UoG) of rabies virus in ChAT::Cre+/-;
RΦGT+/- mice.
Figure 12 figure supplement 5
Figure 12 figure supplement 5 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 2 TA (yellow)
and PL (dark orange) double injections (UCL) of rabies virus in
ChAT::Cre+/-; RΦGT+/- mice.
Figure 12 figure supplement 6
Figure 12 figure supplement 6 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 4 MG (dark blue)
single injections (UoG) of rabies virus in ChAT::Cre+/-;
RΦGT+/- mice.
Figure 12 figure supplement 7
Figure 12 figure supplement 7 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 3 PL (dark
orange) single injections (UoG) of rabies virus in ChAT::Cre+/-;
RΦGT+/- mice.
Figure 12 figure supplement 8
Figure 12 figure supplement 8 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 3 LG (blue) and
TA (yellow) double injections with low titre rabies virus in
ChAT::Cre+/-; RΦGT+/- mice (UoG).
Figure 12 figure supplement 9
Figure 12 figure supplement 9 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 4 LG (blue)
single injections with low titre rabies virus in ChAT::Cre+/-;
RΦGT+/- mice (UoG).
Figure 12 figure supplement 10
Figure 12 figure supplement 10 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 3 TA (yellow)
single injections with low titre rabies virus in ChAT::Cre+/-;
RΦGT+/- mice (UoG).
Figure 12 figure supplement 11
Figure 12 figure supplement 11 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 4 GS (blue)
single injections with rabies virus in Olig2::Cre +/-; RΦGT+/-
mice (MDC).
Figure 12 figure supplement 12
Figure 12 figure supplement 12 Distribution of motor
neurons (triangles) and premotor interneurons (dots) of 3 TA (yellow)
single injections with rabies virus in Olig2::Cre+/-; RΦGT+/-
mice (MDC).
Figure 12 figure supplement 13
Figure 12 figure supplement 13 Distribution of infected
neurons (primary infected motor neurons or secondary infected
interneurons are not distinguished) of 2 GS (blue) and TA (yellow)
double injections and 2 GS single injections of rabies and AAV-Ef1a-B19G
in wild type mice (Salk).
Figure 12 figure supplement 14
Figure 12 figure supplement 14 Distribution of infected
neurons (primary infected motor neurons or secondary infected
interneurons are not distinguished) of 4 GS (blue) single injections of
rabies and AAV-CAG-Flex-oG in Chat::Cre+/- mice (Salk).
Figure 12 figure supplement 15
Figure 12 figure supplement 15 Distribution of infected
neurons (primary infected motor neurons or secondary infected
interneurons are not distinguished) of 3 TA (yellow) single injections
of rabies and AAV-CAG-Flex-oG in Chat::Cre+/- mice (Salk).
Figure 12 figure supplement 16
Figure 12 figure supplement 16 Distribution of infected
neurons (primary infected motor neurons or secondary infected
interneurons are not distinguished) of 4 GS (blue) and TA (yellow)
double injections of PRV-152 and PRV-614 in wild type mice (Salk).
Figure 13 Supplement 1
